Trans-ancestry epigenome-wide association meta-analysis of DNA methylation with lifetime cannabis use.

Cannabis is widely used worldwide, yet its links to health outcomes are not fully understood. DNA methylation can serve as a mediator to link environmental exposures to health outcomes. We conducted an epigenome-wide association study (EWAS) of peripheral blood-based DNA methylation and lifetime cannabis use (ever vs. never) in a meta-analysis including 9436 participants (7795 European and 1641 African ancestry) from seven cohorts. Accounting for effects of cigarette smoking, our trans-ancestry EWAS meta-analysis revealed four CpG sites significantly associated with lifetime cannabis use at a false discovery rate of 0.05 [Formula: see text]: cg22572071 near gene ADGRF1, cg15280358 in ADAM12, cg00813162 in ACTN1, and cg01101459 near LINC01132. Additionally, our EWAS analysis in participants who never smoked cigarettes identified another epigenome-wide significant CpG site, cg14237301 annotated to APOBR. We used a leave-one-out approach to evaluate methylation scores constructed as a weighted sum of the significant CpGs. The best model can explain 3.79% of the variance in lifetime cannabis use. These findings unravel the DNA methylation changes associated with lifetime cannabis use that are independent of cigarette smoking and may serve as a starting point for further research on the mechanisms through which cannabis exposure impacts health outcomes.

Human milk-associated bacterial communities associate with the infant gut microbiome over the first year of life.

Introduction: Microbial communities inhabiting the human infant gut are important for immune system development and lifelong health. One critical exposure affecting the bacterial colonization of the infant gut is consumption of human milk, which contains diverse microbial communities and prebiotics. We hypothesized that human milk-associated microbial profiles are associated with those of the infant gut. Methods: Maternal-infant dyads enrolled in the New Hampshire Birth Cohort Study (n = 189 dyads) contributed breast milk and infant stool samples collected approximately at 6 weeks, 4 months, 6 months, 9 months, and 12 months postpartum (n = 572 samples). Microbial DNA was extracted from milk and stool and the V4-V5 region of the bacterial 16S rRNA gene was sequenced. Results: Clustering analysis identified three breast milk microbiome types (BMTs), characterized by differences in Streptococcus, Staphylococcus, Pseudomonas, Acinetobacter, and microbial diversity. Four 6-week infant gut microbiome types (6wIGMTs) were identified, differing in abundances of Bifidobacterium, Bacteroides, Clostridium, Streptococcus, and Escherichia/Shigella, while two 12-month IGMTs (12mIGMTs) differed primarily by Bacteroides presence. At 6 weeks, BMT was associated with 6wIGMT (Fisher's exact test value of p = 0.039); this association was strongest among infants delivered by Cesarean section (Fisher's exact test value of p = 0.0028). The strongest correlations between overall breast milk and infant stool microbial community structures were observed when comparing breast milk samples to infant stool samples collected at a subsequent time point, e.g., the 6-week breast milk microbiome associated with the 6-month infant gut microbiome (Mantel test Z-statistic = 0.53, value of p = 0.001). Streptoccous and Veillonella species abundance were correlated in 6-week milk and infant stool, and 4- and 6-month milk Pantoea species were associated with infant stool Lachnospiraceae genera at 9 and 12 months. Discussion: We identified clusters of human milk and infant stool microbial communities that were associated in maternal-infant dyads at 6 weeks of life and found that milk microbial communities were more strongly associated with infant gut microbial communities in infants delivered operatively and after a lag period. These results suggest that milk microbial communities have a long-term effect on the infant gut microbiome both through sharing of microbes and other molecular mechanisms.

Differences in DNA Methylation-Based Age Prediction Within Twin Pairs Discordant for Cancer.

DNA methylation-based age acceleration (DNAmAA) is associated with cancer, with both cancer tissue and blood showing increased DNAmAA. We aimed to investigate whether DNAmAA is associated with cancer risk within twin pairs discordant for cancer, and whether DNAmAA has the potential to serve as a biomarker for such. The study included 47 monozygotic and 48 same-sex-dizygotic cancer-discordant twin pairs from the Finnish Twin Cohort study with blood samples available between 17 and 31 years after the cancer diagnosis. We studied all cancers (95 pairs), then separately breast cancer (24 pairs) and all sites other than breast cancer (71 pairs). DNAmAA was calculated for seven models: Horvath, Horvath intrinsic epigenetic age acceleration, Hannum, Hannum intrinsic epigenetic age acceleration, Hannum extrinsic epigenetic age acceleration, PhenoAge and GrimAge. Within-pair differences in DNAmAA were analyzed by paired t tests and linear regression. Twin pairs sampled before cancer diagnosis did not differ significantly in DNAmAA. However, the within-pair differences in DNAmAA before cancer diagnosis increased significantly the closer the cancer diagnosis was, and this acceleration extended for years after the diagnosis. Pairs sampled after the diagnosis differed for DNAmAA with the Horvath models capturing cancer diagnosis-associated DNAmAA across all three cancer groupings. The results suggest that DNAmAA in blood is associated with cancer diagnosis. This may be due to epigenetic alterations in relation to cancer, its treatment or associated lifestyle changes. Based on the current study, the biomarker potential of DNAmAA in blood appears to be limited.

BMI is positively associated with accelerated epigenetic aging in twin pairs discordant for body mass index.

BACKGROUND: Obesity is a heritable complex phenotype that can increase the risk of age-related outcomes. Biological age can be estimated from DNA methylation (DNAm) using various "epigenetic clocks." Previous work suggests individuals with elevated weight also display accelerated aging, but results vary by epigenetic clock and population. Here, we utilize the new epigenetic clock GrimAge, which closely correlates with mortality. OBJECTIVES: We aimed to assess the cross-sectional association of body mass index (BMI) with age acceleration in twins to limit confounding by genetics and shared environment. METHODS AND RESULTS: Participants were from the Finnish Twin Cohort (FTC; n = 1424), including monozygotic (MZ) and dizygotic (DZ) twin pairs, and DNAm was measured using the Illumina 450K array. Multivariate linear mixed effects models including MZ and DZ twins showed an accelerated epigenetic age of 1.02 months (p-value = 6.1 × 10-12 ) per one-unit BMI increase. Additionally, heavier twins in a BMI-discordant MZ twin pair (ΔBMI >3 kg/m2 ) had an epigenetic age 5.2 months older than their lighter cotwin (p-value = 0.0074). We also found a positive association between log (homeostatic model assessment of insulin resistance) and age acceleration, confirmed by a meta-analysis of the FTC and two other Finnish cohorts (overall effect = 0.45 years, p-value = 4.1 × 10-25 ) from adjusted models. CONCLUSION: We identified significant associations of BMI and insulin resistance with age acceleration based on GrimAge, which were not due to genetic effects on BMI and aging. Overall, these results support a role of BMI in aging, potentially in part due to the effects of insulin resistance.

Identical twins carry a persistent epigenetic signature of early genome programming.

Monozygotic (MZ) twins and higher-order multiples arise when a zygote splits during pre-implantation stages of development. The mechanisms underpinning this event have remained a mystery. Because MZ twinning rarely runs in families, the leading hypothesis is that it occurs at random. Here, we show that MZ twinning is strongly associated with a stable DNA methylation signature in adult somatic tissues. This signature spans regions near telomeres and centromeres, Polycomb-repressed regions and heterochromatin, genes involved in cell-adhesion, WNT signaling, cell fate, and putative human metastable epialleles. Our study also demonstrates a never-anticipated corollary: because identical twins keep a lifelong molecular signature, we can retrospectively diagnose if a person was conceived as monozygotic twin.

Validation of the Emotional Tone Index for Families (ETIF): A Multi-Informant Measure of Emotional Closeness.

Despite the importance of emotional closeness (EC) in families, few researchers have accurately measured the construct in a systemic way. Additionally, existing measures rely on ratings from one informant, typically the mother, to provide information on closeness within the entire family system. We examined EC in 140 individuals (37 families) using the Emotional Tone Index for Families (ETIF), a novel, multi-informant measure that obtains bidirectional information about EC within every family relationship. The parent identified as most familiar with the family also completed two widely used single-informant measures: The McMaster Family Assessment Device and the Family Adaptability and Cohesion Evaluation Scales, version IV. The ETIF exhibited good test-retest reliability, high internal consistency, and concurrent validity with the single-informant measures. Though the primary respondent scores correlated highly with overall family closeness, results revealed only a modest association between closeness ratings within each dyad and parents rated higher levels of closeness toward their children than children rated closeness toward parents. These findings suggest that ratings from multiple informants provide valuable information about discrepancies in perceived closeness between family members and other complex family dynamics that cannot be captured by single-informant measures. Limitations, future directions, and implications for practice are discussed.

A pesar de la importancia de la cercanía emocional en las familias, pocos investigadores han medido con precisión el constructo de una manera sistémica. Además, las herramientas de medición existentes dependen de las valoraciones de un informante, normalmente la madre, para proporcionar información sobre la cercanía dentro de todo el sistema familiar. Analizamos la cercanía emocional en 140 personas (37 familias) usando el Índice de Tono Emocional para las Familias (ITEF), una herramienta de medición novedosa que obtiene información bidireccional sobre la cercanía emocional dentro de cada relación familiar por parte de varios informantes. El progenitor identificado como el más familiarizado con la familia también completó dos evaluaciones de un solo informante ampliamente utilizadas: el Dispositivo de McMaster de Evaluación Familiar (McMaster Family Assessment Device) y las Escalas de Evaluación de la Cohesión y la Adaptabilidad Familiar (Family Adapatability and Cohesion Evaluation Scales), versión IV. El ITEF demostró buena fiabilidad de prueba-reprueba, una alta coherencia interna y validez simultánea con las evaluaciones de un solo informante. Aunque los puntajes del encuestado principal se correlacionaron en gran medida con la cercanía familiar general, los resultados revelaron solo una asociación modesta entre las valoraciones de cercanía dentro de cada díada, y los padres indicaron niveles más altos de cercanía hacia sus hijos de lo que los hijos lo hicieron hacia sus padres. Estos resultados sugieren que las valoraciones de varios informantes proporcionan información valiosa acerca de las discrepancias en la cercanía percibida entre los miembros de la familia y otra dinámica familiar compleja que no puede captarse mediante las mediciones de un solo informante. Se explican las limitaciones, las futuras direcciones y las implicancias para la práctica.

Prediagnostic breast milk DNA methylation alterations in women who develop breast cancer.

Prior candidate gene studies have shown tumor suppressor DNA methylation in breast milk related with history of breast biopsy, an established risk factor for breast cancer. To further establish the utility of breast milk as a tissue-specific biospecimen for investigations of breast carcinogenesis, we measured genome-wide DNA methylation in breast milk from women with and without a diagnosis of breast cancer in two independent cohorts. DNA methylation was assessed using Illumina HumanMethylation450k in 87 breast milk samples. Through an epigenome-wide association study we explored CpG sites associated with a breast cancer diagnosis in the prospectively collected milk samples from the breast that would develop cancer compared with women without a diagnosis of breast cancer using linear mixed effects models adjusted for history of breast biopsy, age, RefFreeCellMix cell estimates, time of delivery, array chip and subject as random effect. We identified 58 differentially methylated CpG sites associated with a subsequent breast cancer diagnosis (q-value <0.05). Nearly all CpG sites associated with a breast cancer diagnosis were hypomethylated in cases compared with controls and were enriched for CpG islands. In addition, inferred repeat element methylation was lower in breast milk DNA from cases compared to controls, and cases exhibited increased estimated epigenetic mitotic tick rate as well as DNA methylation age compared with controls. Breast milk has utility as a biospecimen for prospective assessment of disease risk, for understanding the underlying molecular basis of breast cancer risk factors and improving primary and secondary prevention of breast cancer.

Microbial Communities in Human Milk Relate to Measures of Maternal Weight.

The process of breastfeeding exposes infants to bioactive substances including a diversity of bacteria from breast milk as well as maternal skin. Knowledge of the character of and variation in these microbial communities, as well as the factors that influence them, is limited. We aimed to identify profiles of breastfeeding-associated microbial communities and their association with maternal and infant factors. Bilateral milk samples were collected from women in the New Hampshire Birth Cohort Study at approximately 6 weeks postpartum without sterilization of the skin in order to capture the infant-relevant exposure. We sequenced the V4-V5 hypervariable region of the bacterial 16S rRNA gene in 155 human milk samples. We used unsupervised clustering (partitioning around medoids) to identify microbial profiles in milk samples, and multinomial logistic regression to test their relation with maternal and infant variables. Associations between alpha diversity and maternal and infant factors were tested with linear models. Four breastfeeding microbiome types (BMTs) were identified, which differed in alpha diversity and in Streptococcus, Staphylococcus, Acinetobacter, and Pseudomonas abundances. Higher maternal pre-pregnancy BMI was associated with increased odds of belonging to BMT1 [OR (95% CI) = 1.13 (1.02, 1.24)] or BMT3 [OR (95% CI) = 1.12 (1.01, 1.25)] compared to BMT2. Independently, increased gestational weight gain was related to reduced odds of membership in BMT1 [OR (95% CI) = 0.66 (0.44, 1.00) per 10 pounds]. Alpha diversity was positively associated with gestational weight gain and negatively associated with postpartum sample collection week. There were no statistically significant associations of breastfeeding microbiota with delivery mode. Our results indicate that the breastfeeding microbiome partitions into four profiles and that its composition and diversity is associated with measures of maternal weight.

Sex-specific associations of infants' gut microbiome with arsenic exposure in a US population.

Arsenic is a ubiquitous environmental toxicant with antimicrobial properties that can be found in food and drinking water. The influence of arsenic exposure on the composition of the human microbiome in US populations remains unknown, particularly during the vulnerable infant period. We investigated the relationship between arsenic exposure and gut microbiome composition in 204 infants prospectively followed as part of the New Hampshire Birth Cohort Study. Infant urine was analyzed for total arsenic concentration using inductively coupled plasma mass spectrometry. Stool microbiome composition was determined using sequencing of the bacterial 16S rRNA gene. Infant urinary arsenic related to gut microbiome composition at 6 weeks of life (p = 0.05, adjusted for infant feeding type and urine specific gravity). Eight genera, six within the phylum Firmicutes, were enriched with higher arsenic exposure. Fifteen genera were negatively associated with urinary arsenic concentration, including Bacteroides and Bifidobacterium. Upon stratification by both sex and feeding method, we found detectable associations among formula-fed males (p = 0.008), but not other groups (p > 0.05 for formula-fed females and for breastfed males and females). Our findings from a US population indicate that even moderate arsenic exposure may have meaningful, sex-specific effects on the gut microbiome during a critical window of infant development.

Maternal diet during pregnancy is related with the infant stool microbiome in a delivery mode-dependent manner.

BACKGROUND: The gut microbiome has an important role in infant health and immune development and may be affected by early-life exposures. Maternal diet may influence the infant gut microbiome through vertical transfer of maternal microbes to infants during vaginal delivery and breastfeeding. We aimed to examine the association of maternal diet during pregnancy with the infant gut microbiome 6 weeks post-delivery in mother-infant dyads enrolled in the New Hampshire Birth Cohort Study. Infant stool samples were collected from 145 infants, and maternal prenatal diet was assessed using a food frequency questionnaire. We used targeted sequencing of the 16S rRNA V4-V5 hypervariable region to characterize infant gut microbiota. To account for differences in baseline and trajectories of infant gut microbial profiles, we stratified analyses by delivery mode. RESULTS: We identified three infant gut microbiome clusters, characterized by increased abundance of Bifidobacterium, Streptococcus and Clostridium, and Bacteroides, respectively, overall and in the vaginally delivered infant stratum. In the analyses stratified to infants born vaginally and adjusted for other potential confounders, maternal fruit intake was associated with infant gut microbial community structure (PERMANOVA, p < 0.05). In multinomial logistic regression analyses, increased fruit intake was associated with an increased odds of belonging to the high Streptococcus/Clostridium group among infants born vaginally (OR (95% CI) = 2.73 (1.36, 5.46)). In infants delivered by Cesarean section, we identified three clusters that differed slightly from vaginally delivered infants, which were characterized by a high abundance of Bifidobacterium, high Clostridium and low Streptococcus and Ruminococcus genera, and high abundance of the family Enterobacteriaceae. Maternal dairy intake was associated with an increased odds of infants belonging to the high Clostridium cluster in infants born by Cesarean section (OR (95% CI) = 2.36 (1.05, 5.30)). Linear models suggested additional associations between maternal diet and infant intestinal microbes in both delivery mode strata. CONCLUSIONS: Our data indicate that maternal diet influences the infant gut microbiome and that these effects differ by delivery mode.

The premature infant gut microbiome during the first 6 weeks of life differs based on gestational maturity at birth.

BACKGROUND: The impact of degree of prematurity at birth on premature infant gut microbiota has not been extensively studied in comparison to term infants in large cohorts. METHODS: To determine the effect of gestational age at birth and postnatal exposures on gut bacterial colonization in infants, we analyzed 65 stool samples from 17 premature infants in the neonatal intensive care unit, as well as 13 samples from 13 mostly moderate-to-late premature infants and 189 samples from 176 term infants in the New Hampshire Birth Cohort Study. Gut colonization patterns were determined with 16S rDNA microbiome profiling. RESULTS: Gut bacterial alpha-diversity differed between premature and term infants at 6 weeks of age, after adjusting for exposures (p = 0.027). Alpha-diversity varied between extremely premature (<28 weeks gestation) and very premature infants (≥28 but <32 weeks, p = 0.011), as well as between extremely and moderate-to-late premature infants (≥32 and <37 weeks, p = 0.004). Newborn antibiotic use among premature infants was associated with lower Bifidobacterium and Bacteroides abundance (p = 0.015 and p = 0.041). CONCLUSION: Gestational age at birth and early antibiotic exposure have significant effects on the premature infant gut microbiota.

Association of Cesarean Delivery and Formula Supplementation With the Intestinal Microbiome of 6-Week-Old Infants.

IMPORTANCE: The intestinal microbiome plays a critical role in infant development, and delivery mode and feeding method (breast milk vs formula) are determinants of its composition. However, the importance of delivery mode beyond the first days of life is unknown, and studies of associations between infant feeding and microbiome composition have been generally limited to comparisons between exclusively breastfed and formula-fed infants, with little consideration given to combination feeding of both breast milk and formula. OBJECTIVE: To examine the associations of delivery mode and feeding method with infant intestinal microbiome composition at approximately 6 weeks of life. DESIGN, SETTING, AND PARTICIPANTS: Prospective observational study of 102 infants followed up as part of a US pregnancy cohort study. EXPOSURES: Delivery mode was abstracted from delivery medical records, and feeding method prior to the time of stool collection was ascertained through detailed questionnaires. MAIN OUTCOMES AND MEASURES: Stool microbiome composition was characterized using next-generation sequencing of the 16S rRNA gene. RESULTS: There were 102 infants (mean gestational age, 39.7 weeks; range, 37.1-41.9 weeks) included in this study, of whom 70 were delivered vaginally and 32 by cesarean delivery. In the first 6 weeks of life, 70 were exclusively breastfed, 26 received combination feeding, and 6 were exclusively formula fed. We identified independent associations between microbial community composition and both delivery mode (P< .001; Q < .001) and feeding method (P = .01; Q < .001). Differences in microbial community composition between vaginally delivered infants and infants delivered by cesarean birth were equivalent to or significantly larger than those between feeding groups (P = .003). Bacterial communities associated with combination feeding were more similar to those associated with exclusive formula feeding than exclusive breastfeeding (P = .002). We identified 6 individual bacterial genera that were differentially abundant between delivery mode and feeding groups. CONCLUSIONS AND RELEVANCE: The infant intestinal microbiome at approximately 6 weeks of age is significantly associated with both delivery mode and feeding method, and the supplementation of breast milk feeding with formula is associated with a microbiome composition that resembles that of infants who are exclusively formula fed. These results may inform feeding choices and shed light on the mechanisms behind the lifelong health consequences of delivery and infant feeding modalities.